Peptide vaccination or immunotherapy is a therapeutic approach which is currently the subject of a great deal of interest in the context of the prevention or treatment of viral or cancer-related pathologies. The principle thereof is based on immunization with peptides which reproduce T epitopes of viral or tumor antigens recognized by cytotoxic T cells (CTLs), in general of the CD8+ type, which play a major role in the elimination of cells expressing these antigens at their surface.
It will be recalled that CTLs do not recognize whole protein antigens, but peptide fragments thereof, generally comprising 8 to 10 amino acids, presented by the class I major histocompatibility complex (MHC I) molecules expressed at the surface of various cells. The presentation of these peptides is the result of a complex process, called “antigen processing”, which involves 3 main steps:                cytosolic degradation of the antigenic proteins by a multienzyme complex called proteasome;        translocation of the peptides derived from this degradation in the endoplasmic reticulum (ER) by the TAP transporters;         association of these peptides with the 2 chains of the MHC I so as to form stable peptide/MHC I complexes which will be exported to the cell surface.        
The peptide/MHC I complexes interact with the corresponding antigen receptors (TCRs) of T lymphocytes. This interaction induces the stimulation of these lymphocytes, and cell division thereof (clonal proliferation), which results in the generation of the effector lymphocytes bearing the same TCR, which will perform the elimination of the aggressor with respect to the antigens, from which the immune response was induced.
During the processing process, a peptide selection takes place, which results in a hierarchy of presentation by the MHC I at the surface of the cell. The representation of the epitopes at the cell surface will depend in particular on the stability of the antigenic protein in the cytosol, on the sites and frequency of the cleavages performed by the proteasome, on the efficiency of translocation in the ER by the TAP transporters, and especially on the ability of the peptides to attach to the various MHC I molecules and to form stable peptide/MHC I complexes.
The peptides which are preferentially presented by the MHC at the end of the processing process constitute immunodominant epitopes, which are the main participants in the CTL response to the native antigens from which they are derived. On the other hand, the peptides which are only weakly presented constitute subdominant/cryptic epitopes which participate only slightly, or not at all, in this response.
It has been proposed to use peptides corresponding to  those presented by the MHC I to induce a protective response, in particular against viral or tumor antigens.
It has thus been shown that vaccines based on immuno-dominant peptides, generally selected on the basis of their strong affinity for MHC I molecules, make it possible to provide antiviral or antitumor protection in many experimental murine models, and more recently in humans [SCHULTZ et al., Proc. Natl. Acad. Sci. USA, 88, 991 (1991); KAST et al., Proc. Natl. Acad. Sci. USA, 88, 2283, (1991); MARCHAND et al., Int. J. Cancer, 80, 219, (1999); ROSENBERG et al., Nature Med., 4, 321, (1998)].
However, it has also recently been shown that vaccination with immunodominant peptides might, in certain cases, prove to be ineffective. Thus, in chronic infection with a virus with a high mutation rate, such as HIV or HBV, the selection pressure imposed by the natural antiviral CTL response promotes the survival of variants having mutated in the sequence of their immunodominant peptides. These variants are no longer recognized by CTLs specific for immunodominant epitopes [KLENERMAN et al., Nature, 369, 403, (1994); BERTOLETTI et al., Nature, 369, 407, (1994); MOSKOPHIDIS and ZINKERNAGEL, J. Virol., 69, 2187, (1995); BORROW et al., Nat. Med., 3, 205, (1997); GOULDER et al., Nat. Med., 3, 212, (1997)].
Also, in the case of tumors expressing, at high levels, proteins which are also expressed in normal tissues, and which constitute “self antigens”, a phenomenon of tolerance can develop. This tolerance concerns mainly the immunodominant epitopes with strong affinity for MHC. Stimulation of the CTL repertoire specific for these epitopes does not therefore appear to be the best approach for obtaining an effective antitumor protection. 
The use of subdominant/cryptic epitopes, with a low affinity for the MHC, has therefore been proposed. In the case of antiviral vaccination, these epitopes, which are not subjected to a selection pressure similar to that of the immunodominant epitopes, can represent useful targets for eliminating wild-type viruses and also variants thereof. In the case of antitumor vaccination, since the low affinity epitopes participate only slightly, or not at all, in establishing tolerance, the repertoire of antitumor CTLs specific for these epitopes might remain available for in vivo recruitment.
In previous studies, the inventors' team has shown [OUKKA et al., J. Immunol., 157, 3039, (1996)] that it is possible to use subdominant/cryptic peptides in antiviral vaccination. They have also observed that the effectiveness of protection induced by subdominant/cryptic epitopes is less than that obtained with vaccination using the dominant peptide, but that it can be increased by making these peptides more immunogenic through increasing their affinity for the MHC I [TOURDOT et al., J. Immunol., 159, 2391, (1997)].
The usual strategy for increasing the immunogenicity of viral or tumor epitopes consists in increasing their affinity for the MHC I and/or the stability of the peptide/MHC I complex via amino acid substitutions. Specifically, it has been observed that the peptides capable of forming a complex with a given MHC allele have in common the presence, at certain positions, of conserved amino acid residues. A specific anchoring motif, involving amino acids called “primary anchoring residues”, has thus been defined for each allele of the MHC I. It has also been shown that residues located  outside the anchoring sites (secondary anchoring residues) may exert a favorable or unfavorable effect on the affinity of the peptide for the MHC; the presence of these secondary anchoring residues makes it possible to explain the existence, within the peptides having the same anchoring motif specific for a given MHC I, of great variability in the binding affinity, and why peptides which do not have the complete primary anchoring motif may be presented by the MHC I molecules and may have a strong affinity for these molecules.
Many teams have thus succeeded in increasing the immunogenicity of peptides identified as potential viral or tumor immunogens, by increasing their affinity for the MHC I. For example, in mice, LIPFORD et al. [Vaccine, 13, 313, (1995)] have shown that substituting D with I at position 2 of the peptide on the epitope 50-57 of the E6.1 Ag of the papilloma virus, presented by the Kb molecule, increases the stability of the complexes formed with the Kb molecule and makes the epitope immunogenic in vivo, the CTLs induced recognizing the cells transformed by the papilloma virus. BRISTOL et al. [J. Immunol., 160, 2433, (1998)] have also shown that replacing the V residue in the C-terminal position of the epitope 4-12 of the mutated Ras p21 oncogene, with I or L, which are the position-9 anchoring amino acids specific for the Kd molecule, makes it possible to induce a specific CTL response in BALB/c mice. HUDRISIER et al. [Mol. Immunol., 32, 895, (1995)] have identified the residues of the peptide SMIENLEYM (SEQ ID NO: 1) which are involved in binding to the Db molecule, and have produced a series of high affinity peptides derived from the sequence X1AIX4NAEAL (SEQ ID NO: 2) in which X1=Y or K and X4=E or K. 
In humans, POGUE et al. [Proc. Natl. Acad. Sci. USA, 92, 8166, (1995)] have substituted amino acids at the various positions of the epitope 767-484 of the HIV-1 virus reverse transcriptase, presented by the HLA A2.1 molecule, and have shown that substitution of the residue at position 1 with Y or F increases the affinity of the peptide and its ability to induce CTLs from PBLs of seropositive donors possessing the HLA A2.1 allele. PARKHURST et al. [J. Immunol., 157, 2539, (1996)] have performed single substitutions at positions 1, 2 or 3, or double substitutions at positions 1 and 2, or 2 and 3, in the epitopes gp100 209, gp100 280 and gp100 154 of the gp100 Ag associated with melanoma, and have shown that the modified epitopes gp100 209 2M and gp100 280 9V have greater affinity than the unmodified epitope. BAKKER et al. [Int. J. Cancer, 70, 302, (1997)] have obtained, by substitution at one of the anchoring positions (position 2) or outside the anchoring positions (position 8), variants of epitope gp100 154 which have greater affinity than the native epitope. SAROBE et al. [J. Clin. Invest., 102, 1239, (1998)] have obtained an immunogenic variant of the epitope C7A of the HCV virus core protein, presented by HLA A2.1. VALMORI et al. [J. Immunol., 160, 1750, (1998)] have obtained a high affinity derivative of the epitope 26-35 of the MART-1 melanoma antigen presented by HLA A2.1.
It therefore appears to be possible to increase the immunogenicity of subdominant/cryptic epitopes in order to use them in immunotherapy. However, this requires the prior identification of these epitopes. Now, this identification remains problematic, precisely because of their poor immunogenicity.
With the aim of remedying this problem, the inventors  have investigated whether it is possible to define general rules for amino acid substitution which would make it possible to increase the affinity and therefore the immunogenicity of the majority of tumor epitopes presented by the MHC (by class I HLA molecules and in particular the HLA A2.1 molecule), regardless of whether they possess the anchoring amino acids specific for this molecule and, in general, independently of their sequence of origin.
The inventors have thus noted that merely substituting the N-terminal amino acid with a tyrosine residue increases, whatever the sequence of the native peptide, the affinity of this peptide for class I HLA molecules, and in particular the HLA A2.1 molecule, and the stability of the peptide/MHC I complex formed, in a proportion which is all the greater, the lower the affinity of the native peptide. They have also observed that the peptides modified in this way keep the antigen specificity of the natural peptides, and become immunogenic and capable of recruiting, in vivo, a CTL repertoire specific for the corresponding native peptide.